The efficacy of two antivenoms against the venom of North American snakes

Toxicon 41 (2003) 357–365 www.elsevier.com/locate/toxicon The efficacy of two antivenoms against the venom of North American snakes Elda E. Sáncheza,c, Jacob A. Galána, John C. Perezb, Alexis Rodrı́guez-Acostac, Peter B. Chased, John C. Péreza,* a Department of Biology, Natural Toxins Research Center (NTRC), Texas A&M University-Kingsville, MSC 158, Kingsville, TX 78363-8202, USA b Conrad Blucher Institute for Surveying and Science, Texas A&M University-Corpus Christi, Corpus Christi, TX 78412, USA c Universidad Central de Venezuela, Instituto de Medicina Tropical, Sección de Inmunoquı́mica, Apartado 47423, Caracas 1041, Venezuela d University of Arizona, Arizona Poison Control Center, 1703 E. Mabel, P.O. Box 210207, Tucson, AZ 85721, USA Received 4 September 2002; accepted 12 October 2002 Abstract Mortality due to snake envenomation is not a major problem in the United States with approximately 8 – 12 deaths per year, but envenomation is a serious problem that can result in functional disability, loss of extremities, and a costly recovery. Physicians encounter different clinical situations with each new snakebite victim because of the geographical variations in snake venoms. The best and most acceptable form of treatment is the use of antivenom; however, it must be administered as soon as possible since it is not so effective at reducing local signs of envenomation such as necrosis. The antivenom in the United States is in short supply, expensive and may not even be the most effective for neutralizing all North American snake venoms. In this study, we tested two antivenoms. The first was a Crotalidae Polyvalent Fab fragment with Ovine origin (FabO) manufactured in London, and the second was Antivipmyn, a Mexican manufactured antivenom that is F(ab0 )2 fragment produced in horse (Fab2H). The efficacy of the two antivenoms was tested with 15 different snake venoms found in North America. Three different assays were used to test the efficacy of the antivenoms, the in vivo serum protection test (ED50), antihemorrhagic and anticoagulant. The Fab2H antivenom was most effective in neutralizing the hemorrhagic activity of 78% of the hemorrhagic venoms used in this study. In the ED50 assay, the Fab2H antivenom was effective in neutralizing all venoms used in this study, while FabO neutralized all but C. m. molossus venom. However, in most cases, FabO required less antivenom than Fab2H antivenom to neutralize three LD50. q 2003 Published by Elsevier Science Ltd. Keywords: Antivenom; Crotalidae polyvalent immune fab (ovine); Antivipmyn; Crotalus; Agkistrodon; Sistrurus; LD50; ED50; Antihemorrhagic; Hemorrhagic; Coagulopathy 1. Introduction There are 44 subspecies of venomous snakes in the United States and their venoms are different. Envenomation with Viperidae snake venoms can be a painful and terrifying experience that generally results in edema, necrosis, hemorrhage, coagulopathy and, in some cases, death. * Corresponding author. Tel.: þ1-361-593-3805; fax: þ 1-361593-3798. E-mail address: kfjcp00@tamuk.edu (J.C. Pérez). Physicians encounter different clinical situations with each new snakebite victim since venoms are extremely complex mixtures of proteins and may vary considerably even within the same species. The best and most acceptable treatment of systemically envenomated humans is antivenom; however, it must be administered as soon as possible since the damage cannot be reversed. This is assuming that the antivenom is polyvalent and is specific for all snake venoms in the area. Anai et al. (2002) reported that hemorrhagic metalloproteinases in addition to causing hemorrhage also play a key role in 0041-0101/03/$ - see front matter q 2003 Published by Elsevier Science Ltd. PII: S 0 0 4 1 - 0 1 0 1 ( 0 2 ) 0 0 3 3 0 - 6 358 E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 spreading toxins into the circulatory system; it is therefore essential for an antivenom to be effective in neutralizing these components in addition to other pathogenic molecules. According to World Health Organization (1981), the most accepted method for determining antivenom efficacy is by using an ED50 assay in mice. However, this assay is timeconsuming, expensive, requires many mice and large quantities of venom. It is also not directly related to clinical envenomation in humans. In this study the ED50 was a measure of the effective dose that will neutralize three LD50 in mice; as such, it is a useful preclinical test of in vivo neutralization potency. The ED50 that is currently used may not be the best way to determine antivenom efficacy. Developing other assays to study the neutralization of individual venom molecules is important and will require more than one assay (Gutiérrez et al., 1990, 1996; Bogarin et al., 2000). The more toxic venoms required higher dilutions to obtain an LD50 and many of the minor components of venom were diluted. Physicians treat patients that have been injected with crude venom that is undiluted; the only method of determining the correct dose of antivenom is by observing the progression of the local symptoms of the bite. In this study, three different assays (ED50, antihemorrhagic and anticoagulation) were used to compare the efficacy of Fab2H and FabO antivenoms against 15 snake venoms (Table 1) found in the United States. The snakes selected are commonly reported to be responsible for bites; and eight of the 15 venoms reported were from species of snakes used in a previous study of FabO antivenom (Consroe et al., 1995). Additional seven venoms were selected to include other snakes distributed in the United States and Canada. Table 1 shows the venoms used in this study and their previously reported LD50 (Tennant, 1997; Tennant and Bartlett, 2000). 2. Methods and material 2.1. Venoms Venom was extracted from snakes maintained at the Natural Toxins Research Center (NTRC) on the average of every 6 weeks. The snakes were allowed to bite into a nylon cloth membrane over a beaker and the venom is immediately transferred to Eppendorff tubes. The venoms are centrifuged for 5 min at 23 8C at 12,800g to remove cellular debris. They are then transferred to labeled vials and stored at 278 8C until lyophilized. Venoms are lyophilized once they reach a volume of 1 ml and venoms of the same species are never mixed. All venoms for this study were provided by the NTRC at Texas A&M University-Kingsville, Kingsville, TX, with the exception of Crotalus adamanteus venom which was purchased from Sigma-Aldrich, Co. Equal mixtures of lyophilized venoms of the same species were pooled for this study when possible (Table 1). The lyophilized venom samples were reconstituted in physiological saline, centrifuged at 500g for 10 min and filtered using a Millipore Millix HV 0.45 mm filter unit prior to use. Many of the snake venoms have been previously characterized by high performance liquid chromatography (HPLC) and electrophoretic (ET) profiles and can be found on the Internet (http://ntri.tamuk.edu/; Perez et al., 2001). The HPLC and ET profiles are useful in determining how different the venoms are. All protein determinations for venoms and antivenoms were done at 280 nm. 2.2. Antivenoms Antivipmyn (Fab2H) is a polyclonal antivenom F(ab0 )2 fragment of equine origin produced by Instituto Bioclon in Mexico. The venoms used to produce the Fab2H were that of Crotalus durissus and Bothrops asper. The second antivenom is an affinity-purified Fab fragment of ovine origin (FabO) produced by Therapeutic Antibodies, Inc., London, England. The snake venoms used to produce FabO were Crotalus atrox (Western Diamondback Rattlesnake), C. adamanteus (Eastern Diamondback Rattlesnake), Crotalus scutulatus scutulatus (Mojave Rattlesnake), and Agkistrodon piscivorus piscivorus (Eastern Cottonmouth). 2.3. Hemorrhagic assay The method of Omori-Satoh et al. (1972) was used to determine the minimal hemorrhagic dose (MHD) for the crude venoms. A series of eight dilutions were made for each snake venom, of which 0.1 ml of each dilution was injected intracutaneously into the depilated backs of rabbits. After 24 h, the rabbit was sacrificed and the skin removed. A caliper was used to measure the hemorrhagic diameter on the skin and the MHD determined. The MHD is defined as the amount of venom protein that causes a 10 mm hemorrhagic spot. 2.4. Antihemorrhagic assay A modified method used by Gutiérrez et al. (1985) was followed. Four hundred microliters of crude venom containing 20 MHD were incubated for 1 h at 25 8C with 400 ml of various concentrations of antivenom. This was done for each hemorrhagic venom. The backs of rabbits were depilated and 0.1 ml of each concentration of antivenom was injected intracutaneously. Concentrations of antivenom were selected which neutralized 50% of one MHD of the venoms shown in Table 2. Separate rabbits were used for each venom. The AHD is defined as the concentration of antivenom that neutralizes 50% of one MHD. Table 1 Information on venomous snakes used in this study Scientific name Common name Avid pit tag numbersa Mean South Carolina, across central Georgia, southern Alabama, Mississippi, Louisiana, Arkansas, eastern Oklahoma and Texas Texas and Oklahoma 7.8 16.7 12.3d –e –e –e Texas, Oklahoma, Louisiana, Arkansas, Mississippi, Alabama, Tennessee, Illinois, Kentucky, and Missouri 4.88 5.82 5.35f Florida, Alabama, and Mississippi 1.60 1.14 1.35d Collected in Big Springs, TX Mexico, California, Arizona, New Mexico, Texas and Oklahoma 4.07 8.42 6.65h 011-522-004 and 010-782-102 Eastern part of the United States 1.14 0.75 0.92i 010-327-277, 010-875-780 and 010-595-578 011-064-286, 010-526-346, 011-087-008, 010-861-812 010-853-289, 010-308-063, 010-851-785, 010-304079, 010-366-256, 010-366-256, 010-852-101, 011526-554, 010-827-522, 011-285-317, 010-307-545, 011-032-076, 011-069-330, 011-298-046, 010-569261, 011-282-279, 010-308-257 and 011-084-537 011-121-360 Eastern part of the United States Mexico, Arizona, New Mexico, and Texas 2.69 5.16 3.80 3.78 3.25h 4.42i Mexico, New Mexico, Arizona, Nevada, California, and Texas 0.13 0.54 0.34h Arizona 2.29 3.80 3.05f Found in southern California in coastal areas and on Santa Catalina Island Eastern Oregon and Washington and into southern Canada. Central part of the United States 4.53 3.18 3.79i 010-517-016, 011-549-564 and 011-283-585 A. c. laticinctusc Broad-banded Copperhead A. piscivorus leucostomac Western Cottonmouth 010-274-819, 010-287-027, 010-534-615, 011-107322, 010-304-080, 011-065-018, 010-780-370, 010607-567 and 011-282-011 011-028-305, 010-526-104, 010-639-884, 010-304092, 010-304-284, 010-310-380, 011-304-043, 011362-056, 010-300-810, 011-546-548, 011-367-016, 010-783-538, 010-307-059 and 010-547-512 Crotalus adamanteusg Eastern Diamondback rattlesnake Western Diamondback rattlesnake Canebrake rattlesnake Timber rattlesnake Blacktail rattlesnake C. h. horridusc C. molossus molossusc C. scutulatus scutulatus type Ac C. s. scutulatus type Bj C. viridis helleric C. v. oreganusc C. v. viridisc Mojave rattlesnake A Mojave rattlesnake B Southern Pacific rattlesnake Northern Pacific rattlesnake Prairie rattlesnake 011-085-061, 010-367-284, 011-084-009, and 010328-029 011-544-327 and 011-277-867 2.0 –e 2.37 2.84k 2.19h (continued on next page) 359 011-322-841, 010-597-530, 010-545-514, 011-048055, 011-323-801, 011-306-780, 011-107-830, 010365-572, 010-877-303, 011-286-825, 010-805-588, 010-863-606, 010-307-597, 011-070-594, 010-362804, 010-848-365, 011-367-784, 011-517-600, 010585-293, 010-368-319 –e E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 Low Southern Copperhead C. h. atricaudatusc LD50b Hi Agkistrodon contortrix contortrixc C. atroxc Geographical locations 360 2.5. Sonoclot analyzer profiles (procoagulant activity and antiprocoagulant activity) j k i h f g e c d a b Western Massasagua The nine digit avid numbers represents an individual specimen. Information on the snake can be obtained on the NTRC database using the avid numbers (http://ntri.tamuk.edu). Previously reported LD50. Venoms supplied by the NTRC from multiple snakes. Tennant (1997). No reported information is available. Tennant (1998). Venom purchased from Sigma-Aldrich, Co. Tennant and Bartlett (2000). Consroe et al. (1992). Venoms supplied by the NTRC from a single specimen. Glenn and Straight (1982). –e –e –e –e –e –e East and south Texas and southern New Mexico. Texas, Oklahoma, Kansas and parts of Nebraska, Iowa and Missouri 011-054-575, 010-365-378, 010-864-852, 010-524802, 011-282-823 010-277-307, 011-034-340, 010-571-797, 010-325-097 Desert Massasagua Sistrurus catenatus edwardsii S. c. tergiminus Low Hi Geographical locations Avid pit tag numbersa Common name Scientific name Table 1 (continued) LD50b Mean E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 A Sonoclot analyzer was used to measure the effects of venoms on clotting human blood and neutralization of venoms by the antivenoms. A glass bead activated test (gbACT þ Kit obtained from Sienco, Inc.) was used to monitor human blood coagulation on a Sonoclotw Coagulation and Platelet Function Analyzer (Sienco, Inc.). Ten percent citrated whole human blood (3.2% sodium citrate solution) was incubated at 37 8C at least 5 min prior to use. A cuvette was placed into the cuvette holder and 13 ml of 0.25 M CaCl2 was added to one side of the cuvette. Twenty-nine micrograms of crude venom were added to the other side of the cuvette. Three-hundred microliters of warm citrated human blood were added to the cuvette. Data acquisition was analyzed with Signature Viewer; software provided by Sienco, Inc. on an IMAC computer. The same experiment was conducted with the addition of Fab2H and FabO antivenoms. Twenty-six microliters containing 2.2 mg of antivenom were incubated with 29 mg of venom for 30 min at room temperature, and the antivenom/venom mixture was added to the cuvette as described previously. Fresh human whole blood was collected every 5 h to insure that platelets would not age. Human whole blood, venom and antivenom controls were used for each experiment. The percent reduction of coagulation was calculated by the following formula: 100% 2 (As 2 Bs)/(Vs 2 Bs) £ 100% ¼ % reduction. As: the antivenom/venom clot signal at 2 min; Bs: baseline signal at 2 min; Vs: venom signal at 2 min. The higher the percent reduction the better the neutralization. 2.6. Lethal dose (LD50) Six groups of eight mice for each venom were housed in cages and observed throughout the quarantine period and experiments. The LD50 of the 15 venoms listed in Table 1 were determined in BALB/c mice. All venoms in Table 1, with the exception of C. adamanteus, were obtained from the NTRC at Texas A & M University-Kingsville; and, those venoms with a double asterisk are from a single specimen. All venoms were lyophilized and stored at 278 8C until used. When possible, all venoms were pooled from the same species covering the entire range of the snakes. Venoms collected from juvenile, adults, and both sexes were included in this study. Venoms were dissolved in physiological saline at the highest concentration of venoms that were used for injection. The highest concentration was approximately four times higher than the mean LD50 found in Table 1. Two-fold serial dilutions using saline were made to obtain five additional concentrations. All solutions during the experiment were stored at 4 8C and warmed to 37 8C just before being injected into mice. The lethal toxicity was determined by injecting 0.2 ml of venom (at various concentrations) into the tail veins of 18 – 20 g female 361 E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 Table 2 MHD for 15 snake venoms and the antihemorrhagic dose (AHD) of two antivenoms Venoma MHDb (mg) Fab2H AHD (mg)c FabO AHD (mg) Ratiod C. adamanteuse C. v. viridis C. v. helleri Sistrurus catenatus tergiminus C. atrox S. c. edwardsii C. h. horridus C. s. scutulatus-B C. m. molossus A. p. leucostoma C. h. atricaudatus C. v. oreganus A. c. laticinctus A. c. contortrix C. s. scutulatus-A 0.3 0.7 2.25 2.4 2.5 3.5 5.6 12.2 12.5 29 37.5 43 67 143 –g 1 (1) 4.4 (3) 3.3 (2) 8.8 (4) 27 (7) 26.6 (6) 4.4(3) 283 (11) 35.4 (8) 70.8 (9) 212 (10) 425 (12) 283 (11) 26.5 (5) 4 (1) 4.4 (2) 13.3 (5) 13.3 (5) 7 (4) 141.7 (8) 6.5 (3) 35.4 (6) 283 (9) 141.7 (8) –f –f –f 70.8 (7) 0.25 1.0 0.25 0.66 3.85 0.19 0.67 7.9 0.12 0.49 0.37 Number in parenthesis indicates the rank order in which the antivenom neutralized the MHD. Values in bold indicate the antivenom that requires less protein for neutralization. a Pooled venom obtained for the NTRC serpentarium. b MHD: the amount of venom protein injected into the back of depilated rabbit causing a 10 mm hemorrhagic spot in diameter. c Antivenoms were at a starting concentration of 8.5 mg/ml. AHD: the amount of antivenom (mg) that neutralizes 50% of 1 MHD of venom. The AHD is calculated by dividing the starting concentration of antivenom by the antihemorrhagic titer that neutralizes 50% of 1 MHD and then multiplying by the amount of volume injected into the back of a depilated rabbit. d Fab2H AHD/FabO AHD. e C. adamanteus venom was purchased from Sigma-Aldrich, Co. f Indicates that the MHD was not neutralized with equal volume of antivenom at a concentration of 8.5 mg/ml. g Venom contains no hemorrhagic activity. BALB/c mice. The injections were administered using a 1 ml syringe fitted with a 30-gauge, 0.5-in. needle. Saline controls were used. The endpoint of lethality of the mice was determined after 48 h. The calculations for the LD50 were generated by a program on the NTRC homepage (http://ntri.tamuk.edu/serp/index.html) which was based on the method developed by Reed and Muench (1938). 2.7. Serum protection test (ED50) For each antivenom concentration, six groups of eight mice were challenged with a mixture of three LD50 of venom. The ED50 for Fab2H and FabO calculated for all 15 venoms are shown in Tables 2 and 3. Six doses of antivenom were used at each level. Stock venom solutions containing 30 LD50 were freshly prepared at 0 8C before being used. For each group of mice, equal volumes of venom and antivenom were mixed and incubated at 37 8C for 30 min. Each mouse was injected with 0.2 ml of venom/antivenom mixture into the tail vein. The mice were observed for 48 h and the percent survival and ED50 was calculated. Saline controls and antivenom controls were used. The calculations for the ED50 were generated by a program on the NTRC homepage (http://ntri.tamuk.edu/serp/index.html) which was based on the method developed by Reed and Muench (1938). 3. Results The minimal hemorrhagic doses for the 15 venoms ranged from 0.3 to 143 mg with the most hemorrhagic venom being C. adamanteus, and the least being C. s. scutulatus (Table 2). Fab2H antivenom neutralized the hemorrhagic activity of all the hemorrhagic venoms, while FabO neutralized 11 out of the 14 hemorrhagic venoms (Table 2). The i.v. LD50 for the 15 venoms ranged from 0.47 to 6.8 mg/kg body weight with C. s. scutulatus type A being the most potent, and A. c. laticinctus was the least potent (Table 3). Fab2H was effective in neutralizing the LD50 of all the venoms used in this study while FabO was effective in neutralizing all the venoms with the exception of C. m. molossus venom. However, in many of the venoms neutralized by FabO antivenom, it was apparent that FabO was 2.1– 6.7 better than Fab2H antivenom (Table 3). In those cases in which Fab2H antivenom neutralized better than FabO, Fab2H antivenom was just 1.1 –3 times better. The venoms of C. adamanteus, C. horridus atricaudatus and C. h. horridus contained fractions that induced rapid coagulation on human blood (Table 5). Fab2H neutralized the fractions of C. h. horridus and C. h. atricaudatus more effectively than FabO. However, FabO was better in 362 E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 Table 3 LD50 and ED50 of 15 snake venoms and two different antivenoms Venoma LD50b R2 Fab2H ED50c FabO ED50c Ratiod C. s. scutulatus type A C. h. horridus C. h. atricuadatus C. v. viridis Sistrurus catenatus edwardsii C. adamanteuse C. v. helleri C. v. oreganus S. c. tergiminus A. p. leucostoma C. m. molossus C. atrox C. s. scutulatus type B A. c. contortrix A. c. laticinctus 0.47 0.53 1.26 1.56 1.7 1.84 1.9 2.1 2.1 2.75 4.84 5.1 5.1 5.2 6.8 0.99 0.99 0.96 1 0.99 1 0.99 1 0.95 0.99 0.99 1 0.64 0.92 1 140.5 (11) 111.6 (8) 58.9 (3) 93.6 (7) 140 (10) 34.9 (1) 46.7 (2) 114.1 (9) 83.1 (4) 186.8 (12) 93.1 (6) 295 (14) 88.4 (5) 331.6 (15) 293 (13) 21 (4) 20.9 (3) 8.9 (1) 17.7 (2) 226 (12) 70 (6) 70 (6) 121 (10) 78.4 (8) 55.2 (5) NP (15) 310 (14) 278 (13) 93.7 (9) 140.5 (11) 6.7 5.3 6.6 5.2 0.6 0.50 0.67 0.94 1.05 3.3 0.95 0.31 3.5 2.1 Number in parenthesis indicates the rank order in which the antivenom neutralized 3 £ LD50. Values in bold indicate which antivenom required less protein for neutralization. NP: antivenom did not protect. a Pooled venom obtained for the NTRC serpentarium. b The LD50 is the concentration of venom (mg/kg body weight) required to kill 50% of the BALB/c mice injected iv with 0.2 ml of the various snake venoms. LD50 was calculated using the LD50 calculator on the NTRC’s homepage at http://ntri.tamuk.edu/cgi-bin/ld50/ld50. c Expressed as mg of antivenom/kg of mouse body weight; ED50 values were determined against 3 £ LD50 of venoms. d ED50 of Fab2H antivenom/ED50 of the FabO antivenom. e C. adamanteus was purchased from Sigma-Aldrich, Co. neutralizing the procoagulant fractions of C. adamanteus venom. 4. Discussion Differences in venom toxicity and the ability of two antivenoms to neutralize 15 North American venoms were compared in this study. Consroe et al. (1992) reported the i.v. LD50 for 14 snake venoms, and in this study, eight of the same venoms were used to determine the LD50 (Table 4). The LD50 in the Consroe et al. (1992) study were similar to the LD50 in this study, but there were few major differences in the toxicity of the venoms. For example, the C. h. horridus venom in this study was 12 times more toxic than the C. h. horridus venom used in the Consroe et al. (1992) (0.53 vs. 6.32). This is a considerable difference in toxicity and could influence the way a patient is treated. The results of the ED50 studies were different. This is not surprising since the source of venom and strains of mice (BALB/c vs. ICR) used were different. In this study, three different snake venoms (C. h. horridus ) were used and all were from the eastern part of the United States. In the Consroe et al. (1992) study the venom was purchased from Jim Glenn, a former herpetologist at the Western Institute of Biomedical Research at the Utah Medical Center (Salt Lake City, UT). No mention was made of the venom composition or its geographical distribution. There are many variables when comparing ED50 but two of the most important are variation in venom composition and variation in the natural resistance of the animals. Snake venoms are heterogeneous mixtures of toxins that are different in both their qualitative and quantitative characteristics. This makes comparison of venoms difficult. Every strain of mice and humans has a different degree of natural resistance to snake venom. It is also difficult to extrapolate from mice to humans since no evidence exists to support similarities in resistance of the two species. Many investigators have demonstrated differences in venom characteristics even within the same species (Glenn et al., 1983; Glenn and Straight, 1978; Minton and Weinstein, 1986; Adame et al., 1990; Ferreira et al., 1992; Furtado et al., 1991; Anderson et al., 1993; Anaya et al., 1992; Aird, 1985; Huang et al., 1992). The most interesting reported case of venom variability is with the Southern Pacific Rattlesnake (C. v. helleri ). Johnson et al. (1987) reported a difference in venom from an individual Southern Pacific Rattlesnake (C. v. helleri ) in which one venom gland was producing white venom and the other gland was producing yellow venom. The yellow venom was more toxic having an LD50 of 1.6 mg/kg body weight and the white venom was not lethal even up to a concentration of 10 mg/kg. It is important to collect venom throughout the geographical range of different species of snakes and include all ages, and both genders when producing antivenoms. An ideal antivenom should protect the most 363 E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 Table 4 Comparison of LD50 and ED50 data of this study and Consroe et al. (1995) Venoma LD50b Consroe LD50c Ratiod FabO ED50b Consroe FabO ED50c Ratioe C. s. scutulatus-type A C. h. horridus C. h. atricuadatus C. adamanteus C. v. helleri C. m. molossus C. atrox A. c. contortrix 0.47 (1) 0.53 (2) 1.26 (3) 1.84 (4) 1.9 (5) 4.84 (6) 5.1 (7) 5.2 (8) 0.17 (1) 6.32 (8) 0.92 (2) 1.35 (3) 3.48 (4) 4.42 (6) 3.79 (5) 4.99 (7) 2.7 0.08 1.3 1.4 0.5 1.1 1.3 1.0 21 (3) 20.9 (2) 8.9 (1) 70 (4) 70 (4) (7)f 310 (6) 93.7 (5) 4.9 (1) 81.2 (6) 12.7 (2) 22.7 (3) 849.8 (8) 217.7 (7) 39.2 (5) 35.9 (4) 4.2 0.25 0.7 3.1 0.08 –g 7.9 2.6 Numbers in parenthesis indicate order of potency. Venoms used by this study and Consroe et al. (1995). b Current study. LD50 is expressed as mg of antivenom/kg of mouse body weight. c Consroe et al. (1995). LD50 is expressed as mg of antivenom/kg of mouse body weight. ED50 is expressed as mg of antivenom/kg of mouse body weight; ED50 values were determined against three LD50 of venoms. d LD50 of this study/LD50 of Consroe et al. (1992). e ED50 of this study/ED50 of Consroe et al. (1995). f Venom was not neutralized by FabO. g Ratio not determined. a sensitive human from all snake venoms without any side effects. At present, only two antivenoms are approved by the Food and Drug Administration (FDA) in the United States. Previous studies have reported FabO antivenom to be more effective than Wyeth’s antivenom (equine origin) in neutralizing the venom-induced lethality in mice (Consroe et al., 1995). The results in this study using animal models show that Fab2H antivenom was more effective in neutralizing the hemorrhagic and procoagulant activity of most of the venoms used in this study (Tables 2 and 5). Hemorrhagic metalloproteinases are known to cause local tissue damage (hemorrhage, edema and necrosis) by degradation of basement membrane and extracellular matrix surrounding capillaries and small vessels (Bjarnason and Fox, 1994; Baramova et al., 1989). It has been reported that the neutralization of venom hemorrhagic metalloproteinases prevents coagulopathy in an animal model (Anai et al., 2002). In their study, coagulation parameters were monitored after subcutaneous injection of crude B. jararaca venom, neutralized venom for JF 1 factor and purified JF I factor. JF I is a hemorrhagic metalloproteinase purified from B. jararaca venom (Maruyama et al., 1992). It was concluded that crude venom induced unclottable blood and fibrinogen consumption. JF I-neutralized venom and purified JF I did not promote coagulopathy. Anai et al. (2002) concluded that hemorrhagic metalloproteinases in B. jararaca venom played an important role in the development of coagulopathy by causing rapid spreading of thrombin-like enzymes and procoagulants from the venom injected site into the systemic circulation. They hypothesized that hemorrhagins diffuse into the tissue and absorb on to vessels by the degradation of the extracellular matrix and vascular basement membrane causing the release of other venom toxins (thrombin-like enzymes and procoagulants) into the circulation causing coagulopathy problems. In light of this information, an effective antivenom would be one that neutralizes hemorrhagins preventing other venom components from escaping into the circulatory system. Fab2H antivenom was effective in neutralizing all the venoms with regards to venom-induced lethality, while FabO was effective in neutralizing all but C. m. molossus venom (Table 3). Fab2H antivenom was moderately more effective in neutralizing the venoms of C. adamanteus, C. m. molossus, C. s. scutulatus type B, C. atrox, C. v. helleri, C. v. oreganus and S. c. edwardsii. FabO antivenom was considerably more effective in neutralizing the venoms of C. s. scutulatus type A, C. h. horridus, C. h. atricaudatus, C. v. viridis, and A. p. leucostoma, A. c. contortrix and A. c. laticinctus(Table 3). These results are not surprising as the FabO was prepared using venoms from the species of Table 5 Neutralization of procoagulant activity of C. adamanteus, C. h. atricaudatus and C. h. horridus anion exchange fractions by two antivenoms C. adamanteus C. h. atricaudatus Antivenoma 10b 11 12 10 11 12 13 10 Fab2H FabO 64c 16 78 89 57 93 95 89 68 84 100 76 91 97 100 85 a C. h. horridus Antivenoms are at a starting concentration of 85 mg/ml. Fraction numbers of three venoms. c % Reduction: 100% 2 (As 2 Bs)/(Vs 2 Bs) £ 100% ¼ % reduction. The higher the percent reduction the better the neutralization. As: the antivenom/venom clot signal at 2 min; Bs: baseline signal at 2 min; Vs: venom signal at 2 min. b 364 E.E. Sánchez et al. / Toxicon 41 (2003) 357–365 snakes from the USA. It is interesting that a high degree of protection was observed with Fab2H antivenom. The Fab2H may be useful especially in view of the shortage of antivenoms currently available for protection against the venom found in the US. The venoms that were most toxic were the easiest to neutralize. If snake venoms are extremely toxic, then a high dilution must be made to bring the concentration of venom to a dose that will kill 50% of the mice. Many of the toxins in venom could be diluted to non-lethal concentrations. Therefore, the ratio of antivenom to toxins would be much higher for the more potent venoms in an ED50. A. c. laticinctus (Broad-banded copperhead) was the least toxic venom (6.8 mg/kg body weight) in our study. It was 14 time less toxic than C. s. scutulatus type A (Mojave Rattlesnake) (Table 3). The A. c. laticinctus venom was not diluted as much as C. s. scutulatus type A venom to obtain an LD50 and A. c. laticinctus venom was one of the more difficult venoms to neutralize (Table 4). On the other hand, C. s. scutulatus type A, a more toxic venom was easier to neutralize. Both antivenoms easily neutralized another highly toxic venom (Tables 2 and 3), C. adamanteus (Eastern Diamondback Rattlesnake). Similar results were reported in Consroe et al. (1995). The results of this study suggest that there is a need to change the way antivenom efficacy is measured. It would seem more appropriate to evaluate antivenom in a manner that is similar to the way a physician treats patient. A more meaningful test would be to inject a fixed amount of antivenom into a mouse and determine the LD50 in protected mice. This procedure would measure the effects of minor components in venom and would be closer to the way a physician treats snakebite victims. Recurrent coagulopathy problems have been reported in patients that have been treated with FabO antivenom (Seifert et al., 1997; Boyer et al., 1999; Bogdan et al., 2000; Dart and McNally, 2001; Yip, 2002; Ruha et al., 2002). Seifert et al. (1997) reported that recurrent coagulopathy is a problem in C. atrox envenomated patients given FabO antivenom. The results of our study suggest that the coagulopathy could be due to FabO’s inability to effectively neutralize the hemorrhagic proteins of the venoms (Table 1). Other studies have suggested a short elimination half-time of the small Fab antivenom fragment (Chippaux and Goyffon, 1998). Although, FabO antivenom neutralized the lethal dose of most of the venoms in this study; however, its ability to neutralize hemorrhagic activity was considerably less than Fab2H antivenom. The neutralization of venom hemorrhagic metalloproteinases may very well help inhibit coagulopathy. Escalante et al. (2000) suggested that the neutralization of venom hemorrhagins by a synthetic matrix metalloproteinase inhibitor may be effective in not only reducing local lesions, but also preventing systemic coagulopathy. Fab2H antivenom produced using unrelated venoms from another geographical region may be of some use in the US because of a reasonable high level of cross-reactivity. Fab2H antivenom was very effective in neutralizing the hemorrhagic activity of all the venoms used in this study (Table 2). Further studies are needed to determine if Fab2H antivenom will eliminate the problem of recurrent coagulopathy. As of today, very few cases if any, of coagulopathy have been reported when Fab2H antivenom was used. In conclusion, both antivenoms are effective in neutralizing the LD50 of North American venoms; however, Fab2H antivenom was more effective in neutralizing the hemorrhagic activity that has been shown to be a link to coagulopathy. Therefore, Fab2H antivenom appears to be an excellent choice in the treatment of snakebite envenomations in the United States and Canada particularly when considering cost and availability. In spite of recurrent coagulopathy when given FabO to envenomated patients, FabO neutralized the lethal dose of all but one of the venoms used in this study. However, further evaluation and research is still needed to address the issue of recurrent coagulopathy in any antivenom administered to patients. Acknowledgements This research was supported in part by the NIH/RIMI (2P20RR11594) and NIH/SCORE (5-S06-GM08107-27) grants. Special thanks to Nora Diaz De Leon, NTRC program coordinator, Lucy Arispe, live animal curator and Marı́a S. Ramı́rez, technician. Thanks to the Instituto Bioclon, Mexico City, Mexico, for their antivenom (Fab2H) contribution, and thanks to the University of Arizona, Arizona Poison Control Center, Tucson, Arizona, for their antivenom (FabO) contribution. 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